This invention relates generally to methods and devices for preparing and optionally analyzing a cellular sample surface. More particularly, the invention relates to the use of nozzleless acoustic ejection to deposit droplets from a reservoir containing an analysis-enhancing fluid to designated sites on a cellular sample surface. The invention is particularly useful in enhancing the compositional analysis of the sample at the designated sites and in the mass spectrometric imaging of tissue surfaces.
Cellular assays are carried out to provide critical information for the understanding of complex cell functions. One commonly employed technique used in cellular assays involves the immobilization of sample cells on a substrate surface and the controlled exposure of the cells to one or more fluids. Particularly when the sample is small, such assays may require the precise and accurate handling of small volumes of fluid.
A number of techniques have been developed in order to meet the need for precise and accurate handling of small volumes of fluids, but most suffer from one drawback or another. For example, capillaries having a small interior channel (e.g., Eppendorf-type capillaries) are often used to transfer fluids from a pool of fluid. Their tips are submerged in the pool in order to draw fluid therefrom. In order to provide sufficient mechanical strength for handling, however, such capillaries must have a large wall thickness as compared to the interior channel diameter. Thus, the physical dimensions of such capillaries limit their fluid-handling capability. In addition, since any wetting of the exterior capillary surface results in fluid waste, the high wall thickness to channel diameter ratio exacerbates fluid waste. Also, the pool has a minimum required volume driven not by the fluid introduced into the capillary but, rather, by the need to immerse the large exterior dimension of the capillary. As a result, the fluid volume required for capillary submersion may be more than an order of magnitude larger than the fluid volume transferred into the capillary.
Mass spectrometry is a well-established analytical technique in which sample molecules are ionized and the resulting ions are sorted by mass-to-charge ratio. Mass spectrometry has been employed for samples that have been prepared as an array of features on a substrate surface. Surface-based mass spectrometry has been used, for example, to analyze single nucleotide polymorphisms in microarray formats. See, e.g., U.S. Pat. No. 6,322,970 to Little et al.
Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionization technique often used for mass spectrometric analysis of large and/or labile biomolecules, such as nucleotidic and peptidic oligomers, polymers, and dendrimers, as well as for analysis of non-biomolecular compounds, such as fullerenes. MALDI is considered a xe2x80x9csoftxe2x80x9d ionizing technique in which both positive and negative ions are produced. The technique involves depositing a small volume of sample fluid containing an analyte on a substrate comprised of a photon-absorbing matrix material selected to enhanced desorption performance. See Karas et al. (1988), xe2x80x9cLaser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons,xe2x80x9d Anal. Chem., 60:2299-2301. The matrix material is usually a crystalline organic acid that absorbs electromagnetic radiation near the wavelength of the laser. When co-crystallized with analyte, the matrix material assists in the ionization and desorption of analyte moieties. The sample fluid typically contains a solvent and the analyte. Once the solvent has been evaporated from the substrate, the analyte remains on the substrate at the location where the sample fluid is deposited. Photons from a laser strike the substrate at the location of the analyte and, as a result, ions and neutral molecules are desorbed from the substrate. MALDI techniques are particularly useful in providing a means for efficiently analyzing a large number of samples. In addition, MALDI is especially useful in the analysis of minute amounts of sample that are provided over a small area of a substrate surface.
Surface Enhanced Laser Desorption Ionization (SELDI) is another example of a surface-based ionization technique that allows for high-throughput mass spectrometry. SELDI uses affinity capture reagents such as antibodies to collect samples from a complex mixture, which allows in situ purification of the analyte followed by conventional MALDI analysis. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a xe2x80x9cprotein chipxe2x80x9d to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, analytes can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing different reactive moieties at different sites on a substrate surface, throughput may be increased.
Recently, mass spectrometry techniques involving laser desorption have been adapted for cellular analysis. U.S. Pat. No. 5,808,300 to Caprioli, for example, describes a method for imaging biological samples with MALDI mass spectrometry. This method allows users to measure the distribution of an element or small molecule in biological specimens, including tissue slices and individual cells. In particular, the method can be used for the specific analysis of peptides in whole cells, e.g., by obtaining signals for peptides and proteins directly from tissues and blots of tissues. In addition, the method has been used to desorb relatively large proteins from tissues and blots of tissues in the molecular weight range beyond about 80 kilodaltons. From such samples, hundreds of peptide and protein peaks can be recorded in the mass spectrum produced from a single laser-ablated site on the sample. When a laser ablates the surface of the sample at multiple sites and the mass spectrum from each site is saved separately, a data array is produced which contains the relative intensity of any mass at each site. An image of the sample surface can then be constructed for any given molecular weight, effectively representing a compositional map of the sample surface.
One important issue to successful MALDI profiling and imaging as described above is the application of a controlled or uniform coating of a mass-spectrometry matrix material to the tissue surface, either as a series of features or as a continuous coating. The ability to closely compare relative abundances of a given protein between two tissues is dependent on the application of matrix in exactly the same way. Most current small-volume dispensing techniques are not reliable for matrix material application as needed, due to limitations in volume or in accuracy of placement.
A number of patents have described the use of acoustic energy in printing. For example, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquid drop emitter that utilizes acoustic principles in ejecting droplets from a body of liquid ink onto a moving document to form characters or bar codes thereon. As described in a number of U.S. patent applications, acoustic ejection provides for highly accurate deposition of minute volumes of fluids on a surface, wherein droplet volumexe2x80x94and thus xe2x80x9cspotxe2x80x9d size on the substrate surfacexe2x80x94can be carefully controlled, and droplets can be precisely directed to particular sites on a substrate surface. See, e.g., U.S. Ser. Nos. 09/669,996 and 09/964,212 for xe2x80x9cAcoustic Ejection of Fluids from a Plurality of Reservoirsxe2x80x9d, inventors Ellson, Foote and Mutz, filed Sep. 25, 2000 and Sep. 25, 2001, respectively, and assigned to Picoliter Inc. (Mountain View, Calif.). In other words, nozzleless fluid delivery provides high fluid-delivery efficiency through accurate and precise droplet placement. Nozzleless fluid ejection also provides a high level of control over ejected droplet size.
While nozzleless fluid ejection has generally been appreciated for ink printing applications, acoustic deposition is a generally unknown technique in the field of cellular analysis. Recently, focused acoustic energy has been used to manipulate cells and engage in cell sorting. See U.S. Ser. Nos. 09/727,391 and 09/999,166, filed Nov. 29, 2001 and Nov. 29, 2001, respectively for xe2x80x9cFocused Acoustic Energy for Ejection Cells from a Fluid,xe2x80x9d inventors, Mutz and Ellson, assigned to Picoliter Inc. (Mountain View, Calif.), and U.S. Ser. Nos. 10/040,926 and 10/033,739, filed Dec. 28, 2001 and Dec. 27, 2001, respectively, for xe2x80x9cFocused Acoustic Ejection Cell Sorting System and Method,xe2x80x9d inventors Mutz, Ellson, and Lee, assigned to Picoliter Inc. (Mountain View Calif.). Thus, there exist opportunities to improve cellular assay and analysis techniques through the use of acoustic ejection.
In a first embodiment, the invention relates to a method for acoustically depositing droplets of a fluid on a surface of a cell sample. The method involves first providing a reservoir containing a fluid and positioning the cell sample surface in droplet-receiving relationship to the reservoir. Once the reservoir and the cell sample surface are appropriately positioned, focused acoustic energy is applied to eject a droplet of the fluid from the reservoir. As a result, the droplet is deposited on the sample surface at a designated site.
In another embodiment, a system is provided for acoustically depositing droplets of a fluid on a surface of a cellular sample. The system includes a reservoir containing a fluid, an acoustic ejector, an acoustic ejector positioning means, a cellular sample having a surface, and a sample positioning means. The acoustic ejector includes an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation generated. The ejector positioning means is adapted for positioning the acoustic ejector in acoustic coupling relationship to the reservoir. The sample positioning means is adapted for positioning the cellular sample surface such that at least one designated site thereon is in droplet-receiving relationship to the reservoir.
For any embodiment of the invention, the cellular sample may comprise cells from a number of sources. For example, the cells may be extracted from any living or nonliving organism, or they may be taken from a cell culture. In some instances, the cellular sample is a tissue taken from a mammal. The surface of the cellular sample may be compositionally uniform or nonuniform.
Similarly, the fluid used in the invention may vary. For example, the fluid may be or include an analysis-enhancing fluid. In some instances, the analysis-enhancing fluid may contain a detectable label that may be fluorescent, magnetic, or radioactive. Other types of detectable labels may be employed as well. For example, the label moiety may be enzymatic in nature. Furthermore, the fluid may be selected to preferentially interact with specific moieties on the sample surface. Such fluids may contain, for example, enzymes, whole cells, cell extracts, peptides, or nucleotides.
When an analysis-enhancing fluid is used, the sample is typically subjected to conditions effective to allow the analysis-enhancing fluid to interact with the sample surface so as to render the sample surface suitable for analysis. Then, the sample may be analyzed at the designated site. Analysis may range from inspection, in order to determine the extent of any interaction between the analysis-enhancing fluid and the sample surface, to mass spectrometric analysis. MALDI and/or SELDI type techniques may be employed in which energy applied to the designated site effects the release of sample molecules from the sample surface for analysis.
Optionally, a plurality of droplets may be ejected from one or more reservoirs and deposited on the sample surface. In some instances, the droplets are deposited on the sample surface at a single designated site. In other instances, the droplets are deposited on the sample surface at different designated sites. As a result, an array of droplets may be formed on the sample surface so that when the composition of the sample is analyzed at each designated site, a compositional map of the cellular sample surface may be produced.
Thus, another embodiment of the invention relates to a method for analyzing a surface of a cellular sample. The method involves: (a) providing a reservoir containing an analysis-enhancing fluid; (b) positioning the cellular sample surface such that a designated site on the cellular sample surface is in droplet-receiving relationship to the fluid containing reservoir, wherein the designated site is one of a plurality of designated sites within an array of such sites; (c) applying focused acoustic energy in a manner effective to eject a droplet of the fluid from the reservoir such that the droplet is deposited on the sample surface at the designated site; (d) repeating step (b) and (c) with each different designated site so that at least one droplet is deposited at each designated site; (e) applying energy to each of the designated sites to effect release of sample molecules therefrom; and (f) analyzing the released sample molecules. Optionally, analysis of the released sample molecules involves producing a compositional map of the cellular sample surface from the results of mass spectrometric analysis.
Furthermore, the invention provides a system for acoustically depositing a fluid on a surface of a cellular sample. The system comprises: a reservoir containing an analyte-enhancing fluid; an acoustic ejector comprising an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation that is generated; an acoustic ejector positioning means for positioning the acoustic ejector in acoustic coupling relationship to the reservoir; a cellular sample having a surface; a sample positioning means for positioning a cellular sample surface such that a plurality of different designated sites within an array of such sites on the cellular sample surface are successively placed in droplet-receiving relationship to the reservoir; an energy-applying means for applying energy to the designated sites to effect release and ionization of sample molecules therefrom; and an analyzing means for analyzing the sample molecules from the designated sites. Optionally, the energy applying means comprises a photon bombarding means such as a laser, for bombarding the designated site with photons; and the analyzing means comprises a mass spectrometer.